Cloning and expression of HTLV-III DNA

ABSTRACT

The determination of the nucleotide sequence of HTLV-III DNA; identification, isolation and expression of HTLV-III sequences which encode immunoreactive polypeptides by recombinant DNA methods and production of viral RNA are disclosed. Such polypeptides can be employed in immunoassays to detect HTLV-III.

This application is a divisional application of U.S. application Ser.No. 06/693,866, filed Jan. 23, 1985, which is a continuation-in-partapplication of U.S. application Ser. No. 06/659,339, filed Oct. 10,1984, abandoned, which is a continuation-in-part application of U.S.application Ser. No. 06/643,306, filed Aug. 22, 1984, now abandoned.

TECHNICAL FIELDS

This invention is in the fields of molecular biology and virology and inparticular relates to human T cell leukemia virus—type III (HTLV-III).

BACKGROUND

The term human T cell leukemia-lymphoma virus (HTLV) refers to a uniquefamily of T cell tropic retroviruses. These viruses play an importantrole in the pathogenesis of certain T cell neoplasms. There arepresently three known types of HTLVs. One subgroup of the family,HTLV-type I (HTLV-I), is linked to the cause of adult T-cellleukemia-lymphoma (ATLL) that occurs in certain regions of Japan, theCaribbean and Africa. HTLV-type II (HTLV-II) has been isolated from apatient with a T-cell variant of hairy cell leukemia. M. Popovic et al.,Detection, Isolation, and Continuous Production of CytopathicRetroviruses (HTLV-III) from Patients with AIDS and Pre-AIDS. Science,224:497-500 (1984).

HTLV-type III (HTLV-III) has been isolated from many patients withacquired immunodeficiency syndrome (AIDS). HTLV-III refers to prototypevirus isolated from AIDS patients. Groups reported to be at greatestrisk for AIDS include homosexual or bisexual males; intravenous drugusers and Haitian immigrants to the United States. Hemophiliacs whoreceive blood products pooled from donors and recipients of multipleblood transfusions are also at risk. Clinical manifestations of AIDSinclude severe, unexplained immune deficiency which generally involves adepletion of helper T lymphocytes. These may be accompanied bymalignancies and infections. The mortality rate for patients with AIDSis high. A less severe form of AIDS also exists, in which there may belymphadenopathy and depressed helper T cell counts; there is not,however, the devastating illness characteristic of full-blown AIDS.There are many individuals, who are classified as having early AIDS(pre-AIDS), who exhibit these signs. It is not now possible to predictwho among them will develop the more serious symptoms.

Much of the evidence implicates HTLV-III as the etiological agent of theinfectious AIDS. First, there is consistent epidemiology; greater than95% of the patients with AIDS have antibodies specific for HTLV-III.Second, there has been reproducible identification and isolation ofvirus in this disease; more than 100 variants of HTLV-III have beenisolated from AIDS patients. Third, there has been transmission of thedisease to normal healthy individuals who received blood transfusionsfrom infected blood donors.

HTLV-III has been shown to share several properties with HTLV-I andHTLV-II but also to be morphologically, biologically and antigenicallydistinguishable. R. C. Gallo et al., Frequent Detection and Isolation ofCytopathic Retroviruses (HTLV-III) from Patients with AIDS and At Riskfor AIDS. Science, 224:500-503. (1984). For example, HTLV-III has beenshown to be antigenically related to HTLV-I and HTLV-II by demonstratingcross-reactivity with antibodies to HTLV-I and HTLV-II core proteins,p24 and p19, and envelope antigens and by nucleic acidcross-hybridization studies with cloned HTLV-I and HTLV-II DNAS.However, unlike HTLV-I and HTLV-II, it lacked the ability to infect andtransform T cells from normal umbilical cord blood and bone marrow invitro, and has the cytopathic effect on infected cells only.

Like the RNA genome of other retroviruses, the RNA genome of HTLV-IIIcontains three genes which encode viral proteins: 1) the gag gene, whichencodes the internal structural (nucleocapsid or core) proteins; 2) thepol gene, which encodes the RNA-directed DNA polymerase (reversetranscriptase); and 3) the env gene, which encodes the envelopeglycoproteins of the virion. In addition, the HTLV-III genome contains aregion designated Px, located between the env gene and the 3′ LTR, whichappears to be involved in functional killing of the virus.

At this time, AIDS is still difficult to diagnose before the onset ofclinical manifestations. There is no method presently available for theprevention of the disease. Treatment of those with AIDS is generally notsuccessful and victims succumb to the devastating effects HTLV-III hason the body.

SUMMARY OF THE INVENTION

This invention is based upon applicant's cloning of HTLV-III DNA inrecombinant/vector host systems capable of expressing immunoreactiveHTLV-III polypeptides. Based on the cloning of HTLV-III DNA in systemswhich express immunoreactive-polypeptides, applicant has developedmethods useful in the diagnosis, treatment and prevention of AIDS.Applicant has developed methods of detecting HTLV-III and antibodiesagainst HTLV-III in body fluids (e.g., blood, saliva, semen), andmethods useful in immunotherapy (e.g., vaccination and passiveimmunization against AIDS). In addition, applicant has developed methodsof making HTLV-III DNA probes and RNA probes useful in detectingHTLV-III in body fluids.

Polypeptides encoded by segments of the HTLV-III genome have beenproduced by these recombinant DNA methods. For example, polypeptidesencoded by three regions of the HTLV-III genome (an env gene sequence,an env-lor gene sequence and a 1.1 Kb EcoRI restriction fragment fromHTLV-III cDNA) have been produced. The polypeptides expressed have beenisolated. These polypeptides are immunoreactive with sera of patientshaving AIDS and with antibodies to HTLV-III and thus are useful inscreening blood and other body fluids for the presence of antibodiesagainst HTLV-III. Applicant's invention therefor provides a method notonly for diagnosing AIDS, but also for preventing the transmission ofthe disease to others through blood or blood components harboringHTLV-III. The latter is particularly valuable in screening donated bloodbefore it is transfused or used to obtain blood components (e.g., FactorVIII for the treatment of hemophilia; Factor IX)

Polypeptides produced by the recombinant DNA methods are employed in theproduction of antibodies, including monoclonal antibodies, against thevirus. Such antibodies form the basis for immunoassay and diagnostictechniques for directly detecting HTLV-III in body fluids such as blood,saliva, semen, etc. Neutralizing antibodies against the virus may beused to passively immunize against the disease.

Applicant's cloning of HTLV-III DNA in such recombinant vector hostsystems also provides the basis for determination of the nucleotidesequence of HTLV-III DNA. The DNA probes are homologous to DNA regionswhich are unique to the HTLV-III genome. DNA probes provide anothermethod of detecting HTLV-III in blood, saliva or other body fluids. RNAprobes which contain regions unique to the HTLV-III genome can also beformed and used for the detection of HTLV-III in body fluids.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a representation of HTLV-III DNA. FIG. 1 a shows sites atwhich the genome is cut by the restriction enzyme SstI and FIG. 1 bshows the fragments of HTLV-III genome produced through the action ofrestriction enzymes Kpn, EcoRI and HindIII.

FIG. 2 is a representation of HTLV-III DNA. FIG. 2 a shows the locationof restriction enzyme sites in the genome and FIG. 2 b shows thelocation in the HTLV-III genome of DNA inserts in open reading frameclones. The (+) and (−) indicate reactivity and lack of reactivity,respectively, of the fusion protein expressed by cells transformed bythe ORF vectors with sera of AIDS patients.

FIG. 3 shows the nucleotide sequence for HTLV-III DNA SEQ ID NO:4 andthe predicted amino acid sequence of the four longest open readingframes SEQ ID NOS:8-11. Restriction enzyme sites are indicated above thenucleotide sequence.

FIG. 4 shows sites at which the genome is cut by the restriction enzymeEcoRI and construction of recombinant plasmids carrying HTLV-III DNA.

FIG. 5 shows the nucleotide sequence of the ompA signal peptide and thepertinent region of recombinant plasmids ompA1-R-6; ompA2-R-7 andompA3-R-3.

FIG. 6 represents the open reading frame expression vector pMR100 havingHTLV-III DNA.

BEST MODE OF CARRYING OUT THE INVENTION

Despite the similarity between HTLV-III and the other members of theHTLV-bovine leukemia virus (BLV) family of viruses, the biology andpathology of HTLV-III differs substantially. For example, relativelylittle homology has been found in the HTLV-III genome when compared withthat of the HTLV-I or -II genome. Infection with HTLV-III often resultsin profound immunosuppression (AIDS), consequent to the depletion of theOKT4(+) cell population. This effect is mirrored by a pronouncedcytopathic, rather than transforming, effect of HTLV-III infection uponthe OKT4(+) cells in lymphocyte cultures in vitro. In contrast,infection with HTLV-I results in a low incidence of T-cell leukemialymphoma (an OKT4(+) cell malignancy). There is evidence for some degreeof immunodeficiency in HTLV-I patients as well. Infection of primarylymphocytes in culture by HTLV-I and -II results in vitro transformationof predominantly OKT4(+) cells. A cytopathic effect of HTLV-I infectionupon lymphocytes is apparent, but the effect is not as pronounced asthat observed for HTLV-III.

HTLV-III also differs from HTLV-I and -II in the extent of infectiousvirion production in vivo and in vitro. High titers of cell free,infectious virions can be obtained from AIDS patient semen and salivaand from the supernatant of cultures infected with HTLV-III. Very few,if any, cell free infectious virions can be recovered from adult T-cellleukemia lymphoma (ATLL) patients or from cultures infected with HTLV-Ior -II.

Envelope glycoprotein is the major antigen recognized by the antiserumof AIDS patients. In this respect, HTLV resembles other retroviruses,for which the envelope glycoprotein is typically the most antigenicviral polypeptide. In addition, the neutralizing antibodies aregenerally directed toward the envelope glycoprotein of the retrovirus.Serum samples from 88 percent to 100 percent of those with AIDS havebeen shown to have antibodies reactive with antigens of HTLV-III; themajor immune reactivity was directed against p41, the presumed envelopeantigen of HTLV-III. Antibodies to core proteins have also beendemonstrated in serum of AIDS patients, but do not appear to be aseffective an indicator of infection as is the presence of antibodies toenvelope antigen.

The p41 antigen of HTLV-III has been difficult to characterize becausethe viral envelope is partially destroyed during the process of virusinactivation and purification. This invention responds to the great needto characterize this antigenic component of the HTLV-III virus and todetermine the existence and identity of other viral antigenic componentsin several ways. It provides products, such as HTLV-III polypeptides,antibodies to the polypeptides and RNA and DNA probes, as well asmethods for their production. These serve as the basis for screening,diagnostic and therapeutic products and methods.

This invention relates to HTLV-III polypeptides which are produced bytranslation of recombinant DNA sequences encoding HTLV-III proteins.Polypeptides which are produced in this way and which are immunoreactivewith serum from AIDS patients or antibodies to HTLV-III are referred toas recombinant DNA-produced immunoreactive HTLV-III polypeptides. Theyinclude, but are not limited to, antigenic HTLV-III core and envelopepolypeptides which are produced by translation of the recombinant DNAsequences specific to the gag and the env DNA sequences encodingHTLV-III core proteins and envelope glycoproteins, respectively. Theyalso include the polypeptides which are produced by translation of therecombinant DNA sequences included in a 1.1 Kb EcoRI restrictionfragment of HTLV-III cDNA and recombinant DNA sequences specific to thesor gene and the Px genes of HTLV-III. The sor DNA sequence is common toreplication competent HTLV-III viruses. The Px genes contain a codingsequence with one large open reading frame (lor), located between theenv gene and the 3′ end of the HTLV-III genome. Both the env DNAsequences and the lor DNA sequences are located within the same openreading frame of the HTLV-III genome and this gene region is accordinglydesignated env-lor.

The polypeptides encoded by these regions of the HTLV III can be used inimmunochemical assays for detecting antibodies against HTLV-III andHTLV-III infection. These methods can assist in diagnosing AIDS. Inaddition, they can also be employed to screen blood before it is usedfor transfusions or for the production of blood components (e.g., FactorVIII for the treatment of hemophilia). Availability of screeningtechniques will reduce the risk of AIDS transmission.

Detection of antibodies reactive with the polypeptides can be carriedout by a number of established methods. For example, an immunoreactiveHTLV III polypeptide can be affixed to a solid phase (such aspolystyrene bead or other solid support). The solid phase is thenincubated with blood sample to be tested for antibody against HTLV-III.After an appropriate incubation period the solid phase and blood sampleare separated. Antibody bound to the solid phase can be detected withlabeled polypeptide or with a labeled antibody against humanimmunoglobulin.

HTLV-III polypeptides can be used in a vaccine useful for prevention ofAIDS. For vaccination against the virus, immunogenic polypeptides whichelicit neutralizing antibody would be employed. The leading candidatesfor use in vaccines are the viral envelop polypeptides.

The polypeptides can also be used to produce antibodies, includingmonoclonal antibodies, against the HTLV-III polypeptides. Theseantibodies can be used in immunochemical assays for direct detection ofthe virus in body fluids (such as blood, saliva and semen). Assaysemploying monoclonal antibody against specific HTLV III antigenicdeterminants will reduce false-positive results thereby improvingaccuracy of assays for the virus. Antibodies against the virus may alsobe useful in immunotherapy. For example, antibodies may be used topassively immunize against the virus.

The methods of producing the polypeptides are also a subject of thisinvention, as are diagnostic methods based on these polypeptides.

This invention also provides methods for the isolation of genes ofHTLV-III which encode immunoreactive polypeptides; identification of thenucleotide sequence of these genes; introduction of DNA sequencesspecific to these viral DNA sequences into appropriate vectors toproduce viral RNA and the formation of DNA probes. These probes arecomprised of sequences specific to HTLV-III DNA and are useful, forexample, for detecting complementary HTLV-III DNA sequences in bodyfluids (e.g., blood).

HTLV-III Polypeptides

Genetic engineering methods are used to isolate segments of HTLV-III DNAwhich encode immunoreactive HTLV-III polypeptides. Among these arepolypeptides which are immunoreactive with serum from AIDS patients orantibodies to HTLV-III. These polypeptides include the core protein, a15 Kd peptide encoded by a 1.1 Kb EcoRI HTLV-III restriction fragment ofHTLV-III DNA and the envelope glycoprotein. These methods are also usedto sequence the fragments which encode the polypeptides. The proviralgenes integrated into host cell DNA are molecularly cloned and thenucleotide sequences of the cloned provirus is determined.

An E. coli expression library of HTLV-III DNA is constructed. TheHTLV-III genome is cloned and cuts are then made in the cloned HTLV-IIIgenome with restriction enzymes to produce DNA fragments. (FIGS. 1 and2) HTLV-III DNA fragments of approximately 200-500 bp are isolated froman agarose gel, end repaired with T₄ polymerase and ligated to linkerDNA. The linker ligated DNA is then treated with a restriction enzyme,purified from agarose gel and cloned in an expression vector. Examplesof the expression vectors used are: OmpA, pIN (A,B and C), lambda pL,T7, lac, Trp, ORF and lambda gt11. In addition, mammalian cell vectorssuch as pSV2apt, pSV2neo, pSVdhfr and VPV vectors, and yeast vectors,such as GALI and GAL10, may be used.

The bacterial vectors contain the lac coding sequences, into whichHTLV-III DNA can be inserted for the generation of B-galactosidasefusion protein. The recombinant vectors are then introduced intobacteria (e.g., E.coli); those cells which take up a vector containingHTLV-III DNA are said to be transformed. The cells are then screened toidentify cells which have been transformed and are expressing the fusionprotein. For example, the bacteria are plated on MacConkey agar platesin order to verify the phenotype of clone. If functional B-galactosidaseis being produced, the colony will appear red.

Bacterial colonies are also screened with RTLV-III DNA probes toidentify clones containing the DNA regions of interest (e.g., HTLV-IIIgag, pol and env DNA sequences). Clones which are positive when screenedwith the DNA probe and positive on the MacConkey agar plates areisolated.

This identification of cells harboring the HTLV-III DNA sequences makesit possible to produce HTLV-III polypeptides which are immunoreactivewith HTLV-III specific antibody. The cells from the selected coloniesare grown in culture under conditions allowing the expression of thehybrid protein. Cell protein is then obtained by means known in the art.For example, the culture can be centrifuged and the resulting cellpellet broken. Polypeptides secreted by the host cell can be obtained(without disruption of the cells) from the cell culture supernatant.

The total cellular protein is analysed by being run on an SDSpolyacrylamide gel electrophoresis. The fusion proteins are identifiedat a position on the gel which contains no other protein. Western blotanalyses are also carried out on the clones which screened positive.Such analyses are performed with serum from AIDS patients, with theresult that it is possible to identify those clones expressing HTLV-IIIB-galactosidase fusion proteins (antigens) that cross-react with theHTLV-III specific antibody.

Lambda₁₀ clones harboring HTLV-III DNA are cloned from the replicatedform of the virus. As the retrovirus is replicating, double stranded DNAis being produced. The cloned HTLV-III DNA is digested with therestriction enzyme SstI. (FIG. 1 a) Because there are two SstIrecognition sites within the LTR of HTLV-III DNA, one LTR region is notpresent in the cloned DNA sequence removed from the lambda₁₀ vector. Asa result, a small (approximately 200 bp) fragment of the HTLV-III DNA ismissing.

The resulting DNA is linearized and fragments are produced by digestingthe linearized genomic DNA spanning the env gene region with restrictionenzymes. For example, fragments are produced using KpnI or EcoRI plusHindIII, as shown in FIG. 1 b. The resulting 2.3 kb KpnI-KpnI fragments;1.0 kbEcoRI-EcoRI fragments and 2.4 kb EcoRI-HindIII fragments areisolated by gel electrophoresis and electroelution. These fragments arerandomly sheared to produce smaller fragments. The fragments thusproduced are separated on an agarose gel and DNA fragments between about200-500 bp are eluted.

The eluted 200-500 bp DNA fragments are end filled through the use of E.coli T₄ polymerase and blunt end ligated into an open reading frameexpression (ORF) vector, such as pMR100. This ligation may occur at theSmaI site of the pMR100 vector, which contains two promoter regions,hybrid coding sequences of lambdaCI gene and lacI-LacZ gene fusionsequence. In the vector, these are out of frame sequences; as a result,the vector is nonproductive. The HTLV-III DNA is inserted into thevector; the correct DNA fragments will correct the reading frame, withthe result that CI-HTLV-III-B-galactosidase fusion proteins areproduced. The expression of the hybrid is under the control of the lacpromoter. Based on the sequence of pMR100, it appears that if a DNAfragment insert cloned into the SmaI site is to generate a proper openreading frame between the lambdaCI gene fragment and the lac-Z fragment,the inserted DNA must not contain any stop codons in the reading frameset by the frame of the lambdaCI gene.

The recombinant pMR100 vectors are then introduced into E. coli. Thebacteria are plated on MacConkey agar plates to verify the phenotype ofthe clone. If functional B-galactosidase is being produced, the colonywill appear red. The colonies are also screened with HTLV-III DNAprobes, for the purpose of identifying those clones containing theinsert. Clones which are positive when screened with the DNA probe andpositive on the MacConkey agar plates are isolated.

The cells from the selected colonies are grown in culture. The cultureis spun down and the cell pellet broken. Total cellular protein isanalysed by being run on an SDS polyacrylamide gel. The fusion proteinsare identified at a position on the gel which contains no other protein.

Western blot analyses are also carried out on the clones which screenedpositive. Sera from AIDS patients are used, thus making it possible toidentify those clones which express the HTLV-III-B-galactosidase fusionproteins that cross-react with the HTLV-III specific antibody. 1000clones were screened by this method; 6 were positive.

Because of the nature of the pMR100 cloning vehicle, a productive DNAinsert should also be expressed as a part of a larger fusionpolypeptide. HTLV-III env gene containing recombinant clones wasidentified by colony hybridization. The production of larger fusionpolypeptides bearing functional B-galactosidase activity was verified byphenotype identification on MacConkey agar plates; by B-galactosidaseenzymatic assays and by analysis on 75% SDS-polyacrylamide gels.Immunoreactivity of the larger protein with antibody to HTLV-III wasassessed by western blot analysis using serum from AIDS patients. Theselarge fusion proteins also reacted with anti-B-galactosidase and anti-CIantiserum. This finding is consistent with the hypothesis that they areproteins of CI-HTLV-III-lacIZ.

The open reading frame insert fragment of HTLV-III is further analyzedby DNA sequencing analysis. Because one of the two BamHI sites flankingthe SmaI cloning site in pMR100 is destroyed in the cloning step,positive clones are digested with restriction enzymes HindIII and C1aIto liberate the inserted HTLV-III DNA fragment. The HTLV-III ORF insertsare isolated from the fusion recombinant and cloned into M13 sequencingcloning vector mp18 and mp19 digested with HindIll and AccI. DNAsequences of the positive ORF clones are then determined.

Fragments of HTLV-III DNA of approximately 200-500 bps are isolated fromagarose gel, end repaired with T₄ polymerase and ligated to EcoRIlinker. The EcoRI linker ligated DNA is then treated with EcoRI,purified from 1% agarose gel, and cloned in an expression vector, lambdagt11. This vector contains lac Z gene coding sequences into which theforeign DNA can be inserted for the generation of B-galactosidase fusionprotein. The expression of the hybrid gene is under the control of lacrepressor. The lac repressor gene, lac I, is carried on a separateplasmid pMC9 in the host cell, E. coli Y1090. AIDS patient serum wasused to probe the lambda gt11 library of HTLV-III genome DNA containing1.5×10⁴ recombinant phage. In a screen of 5000 recombinants, 100independent clones that produced strong signals were isolated. Thepositive recombinant DNA clones were further characterized for theirspecific gene expression. Rabbit hyperimmune serum against P24 was alsoused to identify the gag gene specific clones. Nick-translated DNAprobes of specific HTLV-III gene, specifically the gag gene, env geneand Px gene were used to group the positive immunoreactive clones intospecific gene region.

Recombinant clones that produced strong signals with AIDS serum andcontain insert DNA spanning the HTLV-III gag, pol, sor and env-lor generegions were examined in detail by mapping their insert with restrictionenzymes and DNA sequencing analysis.

Determination of the Nucleotide Sequence of HTLV-III DNA

Genetic engineering methods are used to determine the nucleotidesequence of HTLV-III DNA. One technique that can be used to determinethe sequence is a shotgun/random sequencing method. HTLV-III DNA issheared randomly into fragments of about 300-500 bp in size. Thefragments are cloned, for example, using m13, and the colonies screenedto identify those having an HTLV-III DNA fragment insert. The nucleotidesequence is then generated, with multiple analysis producing overlaps inthe sequence. Both strands of the HTLV-III DNA are sequenced todetermine orientation. Restriction mapping is used to check thesequencing data generated.

The nucleotide sequence of one cloned HTLV-III genome (BH10) is shown inFIG. 3, and SEQ ID NO:4 in which the position of sequences encoding gagprotein p17 and the N-terminus of gag p24 and the C-terminus of gag p15(which overlaps with the N-terminus of the pol protein) are indicated.The open reading frames (ORF) for pol, sor and env-lor are alsoindicated. The sequence of the remaining 182 base pairs of the HTLV-IIIDNA not present in clone BH10 (including a portion of R, U5, the tRNAprimer binding site and a portion of the leader sequence) was derivedfrom clone HXB2 (SEQ ID NO:3). The sequences of two additional clones(BHB and BH5 (SEQ ID NO:6)) are also shown. Restriction enzyme sites arelisted above the nucleotide sequence; sites present in clone BHB but notin clone BH10 are in parentheses. Deletions are noted ([ ]) atnucleotides 251, 254, 5671 and 6987-7001. The nucleotide positions (tothe right of each line) start with the transcriptional initiation site.The amino acid residues are numbered (to the right of each line) for thefour largest open reading frames starting after the precedingtermination codon in each case except gag which is enumerated from thefirst methionine codon. A proposed peptide cleavage site (V) andpossible asparagine-linked glycosylation sites are shown (*) for theenv-lor open reading frame. The sequences in the LTR derived from clonesBH8 and BH10 (SEQ ID NO:6) listed in the beginning of the figure arederived from the 3′-portion of each clone and are assumed to beidentical to those present in the 5′-LTR of the integrated copies ofthese viral genomes.

Recombinant phage clones harboring HTLV-III DNA, designated λBH-10,λBH-5 and λBH-8, were deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md., 20852 on Jul. 30, 1984under ATCC accession numbers 40125, 40126, and 40127, respectively.

Clone HXB2 was derived from a recombinant phage library of XbaI digestedDNA from HTLV-III infected H9 cells cloned in lambdaJ1, H9 cells arehuman leukemic cells infected by a pool of HTLV-III from blood of AIDSpatients, F. Wong-Staal, Nature, 312, November 1984. Cloning vectorclones BH10, BH8, and BH5 (SEQ ID NO:5) were derived from a library ofSstI digested DNA from the Hirt supernatant fraction of HTLV-IIIinfected H9 cells cloned in lambdagtWes.lambdaB. Both libraries werescreened with cDNA probe synthesized from virion RNA using oligo-dT as aprimer. Clones BH8, BH5, and a portion of HXB2 were sequenced asdescribed by Maxam and Gilbert. (1980) Maxam, A. M. and Gilbert, Co.Methods in Enzymology. 65: 499-560. Clone BH10 was sequenced by themethod of Sanger modified by the use of oligonucleotides complementaryto the M13 insert sequence as primers and using Klenow fragment of DNApolymerase I or reverse transcriptase as the polymerase.

Formation of RNA, RNA Probes and DNA Probes Specific to HTLV-III

DNA sequences which are an entire gene or segment of a gene fromHTLV-III are inserted into a vector, such as a T7 vector. In thisembodiment, the vector has the Tceu promoter from the T cell gene 10promoter and DNA sequences encoding eleven amino acids from the T cellgene 10 protein.

The vectors are then used to transform cells, such as E. coli. The T7vector makes use of the T7 polymerase, which catalyzes RNA formation andrecognizes only T7 promoter, which is the site where RNA polymerasebinds for the initiation of transcription. The T7 polymerase does notrecognize E. coli promotus. As a result, if HTLV-III DNA sequences areinserted after the promoter and polymerase genes of the T7 vector, whichrecognizes them to the exclusion of other signals, and a terminator isplaced immediately after the HTLV-III DNA sequences, the T7 vector willdirect manufacture RNA complementary to the HTLV-III DNA insert.

Determination of the nucleotide sequence of HTLV-III DNA also providesthe basis for the formation of DNA probes. Both RNA probes and DNAHTLV-III probes must have a distinctive region of the HTLV-III genome inorder to be useful in detecting HTLV-III in body fluids. There isrelatively little homology between the HTLV-III genome and the HTLV-Iand -II genomes and probes contain regions which are unique to HTLV-III(i.e., not shared with HTLV-I or -II). For example, nucleotide sequencesin the env gene region of HTLV-III can be used.

Either viral RNA or DNA can be used for detecting HTLV-III in, forexample, saliva, which is known to have a very high concentration of thevirus. This can be done, for example, by means of a dot blot, in whichthe saliva sample is denatured, blotted onto paper and then screenedusing either type of probe. If saliva is used s the test fluid,detection of HTLV-III is considerably faster and easier than is the caseif blood is tested.

Production of Monoclonal Antibodies Reactive with HTLV-III Polypeptides

Monoclonal antibodies reactive with HTLV-III polypeptides are producedby antibody-producing cell lines. The antibody-producing cell lines maybe hybrid cell lines commonly known as hybridomas. The hybrid cells areformed by fusion of cells which produce antibody to HTLV-III polypeptideand an immortalizing cell, that is, a cell which imparts long termtissue culture stability on the hybrid cell. In the formation of thehybrid cell lines, the first fusion partner—the antibody-producingcell—can be a spleen cell of an animal immunized against HTLV-IIIpolypeptide. Alternatively, the antibody-producing cell can be isolatedB lymphocyte which produces antibody against an HTLV-III antigen. Thelymphocyte can be obtained from the spleen, peripheral blood, lymphnodes or other tissue. The second fusion partner—the immortal cell—canbe a lymphoblastoid cell or a plasmacytoma cell such as a myeloma cell,itself an antibody-producing cell but also malignant.

Murine hybridomas which produce monoclonal antibodies against HTLV-IIIpolypeptide are formed by the fusion of mouse myeloma cells and spleencells from mice immunized against the polypeptide. To immunize the mice,a variety of different immunization protocols may be followed. Forinstance mice may receive primary and boosting immunizations of thepurified polypeptide. The fusions are accomplished by standardprocedures. Kohler and Milstein, (1975) Nature (London) 256, 495-497;Kennet, R., (1980) in Monoclonal Antibodies (Kennet et al., Eds. pp.365-367, Plenum Press, NY).

The hybridomas are then screened for production of antibody reactivewith the polypeptide. This can be performed by screening proceduresknown in the art.

Another way of forming the antibody-producing cell line is bytransformation of antibody-producing cells. For example, a B lymphocyteobtained from an animal immunized against HTLV-III polypeptide may beinfected and transformed with a virus such as the Epstein-Barr virus inthe case of human B lymphocytes to give an immortal antibody-producingcell. See, e.g., Kozbor and Rodor (1983) Immunology Today 4(3), 72-79.Alternatively, the B lymphocyte may be transformed by a transforminggene or transforming gene product.

The monoclonal antibodies against HTLV-III polypeptide can be producedin large quantities by injecting antibody-producing hybridomas into theperitoneal cavity of mice and, after an appropriate time, harvesting theascites fluid which contains very high titer of homogenous antibody andisolating the monoclonal antibodies therefrom. Xenogeneic hybridomasshould be injected into irradiated or athymic nude mice. Alternatively,the antibodies may be produced by culturing cells which produce HTLV-IIIpolypeptide in vitro and isolating secreted monoclonal antibodies fromthe cell culture medium. The antibodies produced according to thesemethods can be used in diagnostic assays (e.g., detecting HTLV-III inbody fluids) and in passive immunotherapy. The antibodies reactive withHTLV-III polypeptides provide the basis for diagnostic tests for thedetection of AIDS or the presence of HTLV-III in biological fluids(e.g., blood, semen, saliva) and for passive immunotherapy. For example,it is possible to produce anti p 41, to attach it to a solid phase usingconventional techniques and to contact the body fluid to be tested withthe immobilized antibody. In this way, HTLV-III (antigen) can bedetected in the body fluid; this method results in far fewer falsepositive test results than do tests in which antibody against HTLV-VIIIis detected.

This invention will now be further illustrated by the followingexamples.

EXAMPLE 1 Preparation of Sonicated DNA Fragments

10 μg of gel purified HTLV-III restriction fragments were sonicated tofragment size on average of 500 bps. After sonication, the DNA waspassed through a DEAE-cellulose column in 0.1×TBE in order to reduce thevolume. The DEAE-bound DNA was washed with 5 ml of 0.2 M NaCl-TE (2 MNaCl, 10 mm Tris HCl pH 7.5, 1 mM EDTA) and then eluted with 1 MNaCl-TE, and ethanol precipitated. The size range of the sonicated DNAwas then determined on 1.2% agarose gel. DNA fragments of desired length(200-500 bps) was eluted from the gel. T4 DNA polymerase was used tofill in and/or trim the single strand DNA termini generated by thesonication procedure. DNA fragments were incubated with T4 polymerase inthe absence of added nucleotides for five minutes at 37° C. to removenucleotides from the 3′ end and then all 4 nucleotide precursors wereadded to a final concentration of 100 uM and the reaction mixture wasincubated another 30 minutes to repair the 5′-end single strandedoverhang. The reaction was stopped by heat inactivation of the enzyme at68° C. for 10 minutes. DNA was phenol extracted once, ethanolprecipitated and resuspended in TE.

EXAMPLE 2 Cloning of Random Sheared DNA Fragments

The sonicated blunt end repaired HTLV-III DNA fragments were ligatedinto the SmaI site of the ORF expression vector pMR100 and transformedinto host cell LG90 using standard transformation procedures.B-galactosidase positive phenotype of the transformant were identifiedby plating the transformed cell on ampicillin (25 μg/ml) containingMcConkey agar plates and scoring the phenotype after 20 hours at 37° C.

EXAMPLE 3 Hybrid Protein Analysis

Ten milliliter samples of cells from an overnight saturated culturegrown in L broth containing ampicillin (25 μg/ml) were centrifuged, thecell pellet was resuspended in 500 μl of 1.2 fold concentrated Laemmlisample buffer. The cells were resuspended by vortexing and boiling for 3minutes at 100° C. The lysate was then repeated by being forced througha 22 gauge needle to reduce the lysate viscosity. Approximately 10 μl ofthe protein samples were electrophoresed in 7.5% SDS-PAGE(SDS-polyacrylamide) gels.

Electrophoretic transfer of proteins from SDS-PAGE gels tonitrocellulose paper was carried out according to Towbin et. al. Afterthe transfer, the filter was incubated at 37° C. for two hours in asolution of 5% (w/v) nonfat milk in PBS containing 0.1% antifoam A and0.0001% merthiolate to saturate all available protein binding sites.Reactions with AIDS antisera were carried out in the same milk buffercontaining 1% AIDS patient antisera that had been preabsorbed with E.coli lysate. Reactions were performed in a sealed plastic bag at 4° C.for 18-24 hours on a rotatory shaker. Following this incubation, thefilter was washed three times for 20 minutes each at room temperature ina solution containing 0.5% deoxycholic, 0.1 M NaCl, 0.5% triton X-100,10 mm phosphate buffer pH 7.5 and 0.1 mM PMSF.

To visualize antigen-antibody interactions, the nitrocellulose was thenincubated with the second goat antihuman antibody that had beeniodinated with ¹²⁵I. The reaction with the iodinated antibody wascarried out at room temperature for 30 minutes in the same milk bufferas was used for the first antibody. The nitrocellulose was then washedas previously described and exposed at −70° C. using Kodak XAR5 filmwith an intensifying screen.

EXAMPLE 4 Screening of the HTLV-III ORF Library by Colony Hybridization

E. coli LG90 transformants were screened with HTLV-III DNA probescontaining the DNA regions of interest (e.g. HTLV-III gag, env or Pxgene specific sequences). Colonies were grown on nitrocellulose filtersand screened according to the procedure of Grunstein and Hogness byusing a nick-translated HTLV-III DNA as hybridization probe.

The DNA fragment was in general excised by restriction endonucleasedigestion, gel purified, and ³²P-labeled to a specific activity of0.5×10⁸ cpm/μg by nick-translation (Rigby, P. W. J. et al., J. Mol.Biol. 113, 237 (1977). Duplicate nitrocellulose filters with DNA fixedto them were prehybridized with 6×SSC (0.9 M NaCl/0.09 M sodium citrate,pH 7.0), 5×Denhardt's solution (Denhardt's solution: 0.02% each ofpolyvinylpyrrolidone, Ficoll and bovine serum albumin) 10 μg ofdenatured sonicated E. coli DNA per ml at 55° C. for 3-5 hours. Thefilters were then placed in a fresh sample of the same solution to whichthe denatured hybridization probe had been added. Hybridization waspermitted to take place at 68° C. for 16 hours. The filters were washedrepeatedly in 0.3×SSC at 55° C., and then exposed to x-ray film.

EXAMPLE 5 Recombinant DNA Produced Peptide of HTLV-III which isImmunoreactive with SERA from Patients with AIDS

An expression vector, pIN-III-ompA (ompA) was used. ompA has thelipoprotein (the most abundant protein in E.coli) gene promoter (1pp)and the lacUV5 promoter-operator (FIG. 1). ompA vectors also contain theDNA segment encoding the lac repressor, which allows the expression ofthe inserted DNA to be regulated by lac operon inducers such as IPTG.The ompA cloning vehicles contain three unique restriction enzyme sitesEcoRI, HindIII, Bam HI in all three reading frames and permit theinsertion of DNA into any of these restriction sites.

Various restriction fragments were excised from the recombinant clone,lambdaBH10, which contains a 9 Kb long HTLV-III DNA insert in the SstIsite of the vector lambdagtWES lambdaB. These restriction fragments werethem inserted into the ompA vectors at all three reading frames and usedto transform E.coli JA221 cells. Transformants were first screened forHTLV-III DNA by in situ colony hybridization using nick-translatedHTLV-III DNA probes. The positive clones were then screened forexpression of HTLV-III antigenic peptides using HTLV-III specificantibodies. For this, lysates of E.coli cell containing HTLV-III DNArecombinant plasmids were electrophoresed on 12.5% SDS-polyacrylamidegel and electroblotted onto nitrocellulose filters. The filters werethen incubated first with well-characterized sera from AIDS patients andnext with ¹²⁵I-labelled goat anti-human IgG antibodies. The washedfilters were autoradiographed to identify peptides reactive withanti-HTLV-III antibodies.

Several gene segments that encode peptides showing immunoreactivity withanti-HTLV-III antibodies were demonstrated. Among these is a 1.1 KbEcoRI restriction fragment. This fragment was inserted into ompA vectorsin all three reading frames (FIG. 4). Cells were grown at 37° C. in Lbroth containing 100 μg/ml. ampicillin to an OD₆₀₀ of 0.2. At this time,the cell cultures were divided into two aliquots. IPTG was added to onealiquot to a final concentration of 2 mM (induced). IPTG was not addedto the other aliquot (uninduced). Upon IPTG induction, transformants ofall three plasmid constructs (designated OmpA₁-R-6 (O1R6), OmpA₂-R-7(O2R7), and OmpA₃-R-3 (O3R3)) produced a 15 Kd peptide that is stronglyreactive with anti-HTLV-III antibodies in sera from AIDS patients Thisreactivity is not detected when sera from normal individuals is used.

DNA sequence data of the HTLV-III genome indicates that there is an openreading frame inside the pol gene located at the 5′-end of the EcoRIfragment. DNA sequence analysis of the three recombinant constructs,O1R6, O2R7 and P3R3, confirmed that each of these recombinants has adifferent reading frame of the HTLV-III plus strand coupled to thecoding sequence of each vector. Only in O3R3 is the reading frame of theinserted DNA in phase with that set by the signal peptide in the ompAvector; in O1R6 and O2R7 the pol gene segment DNA is out of phase (FIG.5).

There is a 6 bp ribosome binding site, AAGGAG (Shine-Dalgarno sequence),located at nucleotide position 24-29 and an initiation codon, ATG,located 11 bp downstream (position 41-43). The 15 Kd peptide synthesizedby all three recombinants appears to be translated from the transcriptsusing this internal initiation codon. If this is true, the peptidestarts from the ATG located at position 41-43 and ends at the stop codonat position 446-448, producing a peptide of 135 amino acid residuesencoded by the 3′-end segment of the pol gene of HTLV-III.

In addition to the 15 Kd peptide, the O3R3 construct, in which thereading frame of the HTLV-III DNA pol gene is in phase with that set bythe vector, produced two additional peptides about 19 Kd and 16.5 Kd insize. It is possible that the 19 Kd peptide contains an additional 35amino acid residues, 21 of which are from the signal peptide encoded bythe ompA₃ vector and 14 encoded by the inserted HTLV-III DNA itself. The16.5 Kd peptide may be the processed 19 Kd peptide in which the signalpeptide is cleaved.

The O1R6 and O2R7 constructs also produce another peptide of about 17.5Kd and weakly reactive with sera of AIDS patients. The origin of thepeptide is not clear. The 1.1 Kb EcoRI fragment contains a secondpotential coding region designated as the short open reading frame (SOR)extending from nucleotide position 360 to 965 (FIG. 4). Four of the fiveAUG methionine codons in this region are near the 5′-end of this openreading frame. This DNA segment could encode peptides of 192, 185, 177or 164 amino acid residues. However, there is no clearly recognizableribosome binding site at the 5′-end of this open reading frame.

Further evidence also supports the conclusion that the 15 Kd peptide isindeed derived from the pol gene. First, deletion of the 3′-end StuI toEcoRI fragment from the 1.1 Kb EcoRI insert from O1R6, O2R7 and O3R8(FIG. 4) does not affect the synthesis of the 15 Kd peptide. Second,clones containing only the 5′-end EcoRI to NdeI fragment still producethe same 16 Kd peptide. Finally, several recombinant clones containingvarious DNA fragments having the SOR coding sequence properly insertedinto the open reading frame cloning vector, pMR100, producedlambdaCI-HTLV-III B-galactosidase tripartite fusion proteins which havevery little immunoreactivity with anti-HTLV-III antibodies present insera from AIDS patients.

Significant immunoreactivity against the 15 Kd peptide derived from theviral pol gene in sera from AIDS patients was detected. The identity ofthis immunoreactive peptide, with respect to the banding pattern ofHTLV-III virion antigen in SDS-polyacrylamide gel electrophoresis, wasdetermined by means of a competition inhibition immunoassay. PurifiedHTLV-III virions were treated with SDS, electrophoresed, andelectroblotted onto a nitrocellulose filter. Identical filter stripscontaining disrupted HTLV-III virions were incubated with wellcharacterized serum from an AIDS patient in the presence or absence oflysates of O1R6, O2R7, or control bacterial clones. The specificimmunoreaction between anti-HTLV-III antibodies present in sera of theAIDS patients and the blotted virion proteins were then revealed by¹²⁵I-labeled goat anti-human antibody. Lysates of O1R6 block theimmunoreactivity of the viral p31 protein with the AIDS serum, whilelysates of control cells do not. This result suggests that therecombinant 16 Kd peptide encoded by 3′-end of the viral pol gene isalso a part of another virion protein, p31, in contrast to the viewshared by some that p31 is a cellular protein which co-purifies withHTLV-III virions.

The prevalence in the sera of AIDS patients of antibodies against the 15Kd peptide was also evaluated. In Western blot analysis employing thelysate of O1R6 as the source of antigen, a panel of coded sera from AIDSpatients and normal healthy individuals was tested. All of the 20 AIDSsera and none of the 8 normal controls reacted with the 15 Kd peptide.These data indicate that most, if not all, AIDS patients produceantibodies against the viral p31 protein.

EXAMPLE 6 Expression in E. coli of Open Reading Frame Gene Segments ofHTLV-III

HTLV-III DNA was excised from lambda BH-10, which is a previouslyconstructed recombinant lambda phage containing a 9 Kb segment ofHTLV-III DNA inserted into the vector lambdagtwes lambda B (FIG. 2 a).This HTLV-III DNA was sonicated and DNA fragments of about 0.5 Kbpurified by gel electrophoresis, end repaired, and inserted into theSmaI site of the open reading frame (ORF) vector, pMR100 (FIG. 6). Thisvector contains a bacterial lac promoter DNA segment linked to a secondDNA fragment containing a hybrid coding sequence in which the N-terminus(5′ segment) of the lambda CI gene of bacteriophage lambda is fused toan N-terminal-deleted lacIZ gene (3′ segment). A short linker DNAfragment, containing a SmaI cloning site, has been inserted betweenthese two fragments in such a manner that a frame shift mutation hasbeen introduced upstream of the lacIZ-coding DNA. As a result, pMR100does not produce any detectable B-galactosidase activity when introducedinto cells of the Lac⁻ host E. coli LG90. The insertion of foreign DNAcontaining an open reading frame, in this case the HTLV-III DNA, at theSmaI cloning site can reverse the frame shift mutation if the insertedcoding sequence is in the correct reading frame with respect to both thelambdaCI leader and the lacIZ gene. Transformants were screened onMacConkey plates to detect individual clones that expressedβ-galactosidase enzymatic activity in situ.

Among the 6000 ampicillin resistant transformants screened, about 300were found to express B-galactosidase activity. Colony hybridizationusing ³²p-labelled nick-translated HTLV-III DNA as a probe revealed thatall these Lac⁺ clones contained HTLV-III DNA. In the Lac⁺ clones theHTLV-III fragment inserted into the Sma I site of pMR100 must contain nostop codons in the reading frame set by the lambdaCI leader segment andthe lacIZ gene must also be in the correct translational reading frame.The three-element-fused genes were expressed as tripartite fusionproteins, having a portion of the lambdaCI protein at the N-terminus,the HTLV-III segment in the middle, and the lacIZ polypeptide at theC-terminus.

The proteins produced by the Lac⁺ clones were analyzed by resolving celllysates on 7.5% SDS-polyacrylamide gels along with those of the controlLac⁺ clone pMR200, which produced a lambdaCI-β-galactosidase fusionprotein. The lacIZ gene in pMR200 is identical to that in pMR100 exceptthat it has a single base pair deletion which brings it in phase withthe lambdaCI gene to produce an active β-galactosidase. By virtue of thevery large size of the β-galactosidase and its fusion proteins, they areseparated from the bulk of proteins in the cell lysates on theSDS-polyacrylamide gels and can be easily identified by Coomassiebrilliant blue staining. Some of the Lac⁺ clones containing HTLV-III DNAproduce polypeptides that are larger (15,000 to 27,000 daltons) than thelambdaCI-lacIZ fusion protein. These findings are consistent with datathat the DNA inserts are up to 700 bp long. The β-galactosidase fusionproteins accounted for about 1-2% of total cellular protein.

The peptides produced by the Lac⁺ clones were examined by Western blotanalysis for immunoreactivity with sera from AIDS patients. After thelysates of Lac⁺ clones were electrophoresed in SDS-polyacrylamide gels,they were electro-transferred to nitrocellulose filters. These proteinblots were first reacted with AIDS patient sera and then with¹²⁵I-labeled goat anti-human IgG. The recombinant peptides also reactedwith anti-B-galactosidase antiserum, consistent with the propositionthat they had the general structure lambdaCI-HTLV-III peptide-LacIZ.From the immunoreactivity pattern of the negative controls, pMR100 andpMR200, which do not contain an HTLV-III DNA insert, it is evident thatthis particular AIDS serum contains antibodies reactive with severalbacterial proteins of the host E. coli. This is not surprising, sinceAIDS patients are usually infected with a number of bacteria. AbsorbingAIDS patient sera with Sepharose 4B conjugated with E. coli extractreduced the background immunoreactivity to some extent but did notcompletely eliminate it.

About 300 independent HTLV-III DNA-containing Lac⁺ colonies wereanalyzed in SDS polyacrylamide gels using Coomassie brilliant bluestaining and Western blotting. About half of them were found to expressfusion proteins containing extra peptides of about 100-200 amino acids,corresponding to DNA inserts of 300-600 bp long. Of these fusionproteins, 20 were found to react specifically with sera from AIDSpatients. The unreactive clones probably contain peptides that fold insuch a way that they are not reactive with antibodies or correspond toregions of HTLV-III protein molecules which are not immunogenic in AIDSpatients. The other half of the Lac⁺ clones expressed fusion proteinswhose sizes were not obviously different from that of the lambdaCIB-galactosidase protein. None from this group of fusion proteins wasfound to react with sera from AIDS patients.

The HTLV-III DNA inserts from Lac⁺ ORF clones were mapped to specificsegments in the HTLV-III genome using Southern blotting procedures. Inthese studies, each plasmid clone was labelled with ³²P bynick-translation and hybridized to a battery of HTLV-III DNA restrictionfragments. This hybridization analysis mapped all of the Lac⁺ ORF clonesinto four open reading frame segments designated ORF-A, ORF-B, ORF-C,and ORF-D (FIG. 2 a) consistent with the DNA sequencing data. The openreading frames ORF-A and -B, corresponding to the coding regions of thegag and pol genes, are 1.5 Kb and 3.0 Kb long, respectively. ORF-C isabout 0.6 Kb long, slightly overlaps with the ORF-B region, and iscapable of encoding a polypeptide of 21 overlaps with the ORF-B region,and is capable of encoding a polypeptide of 21 Kd. The location of ORF-Cand its overlap with the pol gene are reminiscent of the structure ofthe env genes in HTLV-I and -II. However, ORF-C, designated as the shortopen reading frame (sor), is too short to code for the entire envelopeprotein. The fourth open reading frame, ORF-D, is 2.5 Kb long and couldencode both a large precursor of the major envelope glycoprotein andanother protein derived from the 3′ terminus, which may be analogous tothe lor products of HTLV-I and -II. This gene region of HTLV-III,designated env-lor, is at least twice as long as the lor of HTLV-I andHTLV-II and it is presently unclear whether single or multiple proteinsare encoded herein.

Both Southern blotting and DNA sequencing studies were employed toanalyze a number of clones. As shown in FIG. 2 b, the Lac⁺ ORF clonesexpressing fusion proteins immunoreactive with sera from AIDS patientswere located in ORF-A (e.g. #175 and #191), ORF-B (e.g. #13, 31, and162), or ORF-D (e.g. #113, 121, and 127) and not in the sor region. Notall peptides in these regions were immunoreactive, e.g. ORF clone #76located in ORF-D.

Analysis of the open reading frame structures in HTLV-III posedquestions as to which open reading frame(s) corresponds to the env gene.It is possible that the env-lor region in HTLV-III contains all or apart of the env gene in addition to the presumed lor gene. Recentevidence suggests that the lor in HTLV-I encodes a 42 Kd proteininvolved in the process of viral activation and transformation. When thelysate of one of the ORF clones (#127 in FIG. 2 b) was tested againstsera from 20 AIDS patients and 12 healthy normals in a stripradioimmunoassay based on the Western blot technique, immunoreactivityagainst the lambdaCI-HTLV-III-B-galactosidase fusion polypeptide wasdetected in the sera from 19 of the AIDS patients and none from normalcontrols. This result indicates that the protein encoded by the portionof the env-lor region contained in ORF clone #127 is produced inHTLV-III infected cells and induces antibody production in most if notall AIDS patients.

Industrial Applicability

This invention has industrial applicability in screening for thepresence of HTLV-III DNA in body fluids and the diagnosis of AIDS.

Equivalents

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention and are coveredby the following claims.

1. An isolated HIV-1 RNA comprising (i) the gag open reading frameconsisting of nucleotides 113-1648 of SEQ ID NO: 4 or nucleotides111-1646 of SEQ ID NO: 5, or (ii) the gag open reading frame encoding,SEQ ID NO:
 8. 2. An isolated HIV-1 RNA comprising (i) the pol openreading frame consisting of nucleotides 1408-4452 of SEQ ID NO: 4 ornucleotides 1406-4450 of SEQ ID NO: 5, or (ii) the pol open readingframe encoding SEQ ID NO:
 9. 3. An isolated HIV-1 RNA comprising (i) thesor open reading frame consisting of nucleotides 4367-4975 of SEQ ID NO:4 or nucleotides 4365-4973 of SEQ ID NO: 5, or (ii) the sor open readingframe encoding SEQ ID NO:
 10. 4. An isolated HIV-1 RNA comprising (i)the env or env-lor open reading frame consisting of nucleotides5560-8148 of SEQ ID NO: 4, or nucleotides 205-2776 of SEQ ID NO: 6, or(ii) the env or env-lor open reading frame encoding SEQ ID NO:
 11. 5. Adiagnostic means comprising said isolated HIV-1 RNA according to claim 1for detecting HIV-1 in body fluids.
 6. A diagnostic means comprisingsaid isolated HIV-1 RNA according to claim 2 for detecting HIV-1 in bodyfluids.
 7. A diagnostic means comprising said isolated HIV-1 RNAaccording to claim 3 for detecting HIV-1 in body fluids.
 8. A diagnosticmeans comprising said isolated HIV-1 RNA according to claim 4 fordetecting HIV-1 in body fluids.
 9. A fragment of the isolated HIV-1 RNAof claim 1 comprising at least 20 contiguous nucleotides of nucleotides113-1648 of SEQ ID NO: 4 or nucleotides 111-1646 of SEQ ID NO: 5 or atleast 200 contiguous nucleotides encoding SEQ ID NO:
 8. 10. A fragmentof the isolated HIV-1 RNA of claim 2 comprising at least 20 contiguousnucleotides of nucleotides 1408-4452 of SEQ ID NO: 4 or nucleotides1406-4450 of SEQ ID NO: 5 or at least 200 contiguous nucleotidesencoding SEQ ID NO:
 9. 11. A fragment of the isolated HIV-1 RNA of claim3 comprising at least 20 contiguous nucleotides of nucleotides 4367-4975of SEQ ID NO: 4 or nucleotides 4365-4973 of SEQ ID NO: 5 or at least 200contiguous nucleotides encoding SEQ ID NO:
 10. 12. A fragment of theisolated HIV-1 RNA of claim 4 comprising at least 20 contiguousnucleotides of nucleotides 5560-8148 of SEQ ID NO: 4 or nucleotides205-2776 of SEQ ID NO: 6 or at least 200 contiguous nucleotides encodingSEQ ID NO:
 11. 13. A diagnostic means comprising the fragment of claim9.
 14. A diagnostic means comprising the fragment of claim
 10. 15. Adiagnostic means comprising the fragment of claim
 11. 16. A diagnosticmeans comprising the fragment of claim 12.